The goals of this bridge proposal focus on the qualitative and quantitative analyses of lipid components in the various subcompartments of macrophages with the intent of furthering our knowledge of how these lipids are involved in the regulation of macrophage-initiated disorders. Macrophages are the hallmark of chronic inflammation. They are recognized as major contributors to the initiation and progress of atherosclerosis, rheumatoid arthritis, and fibrosis. Through major investments in research and development of sophisticated technology to analyze genes and proteins our understanding of the details of how such molecules are involved in macrophage-related disorders has occurred. Unfortunately, this has not been the case for the lipid details and it is the details that are the sights of pharmaceutical intervention. A major objective of this bridge proposal is to build on the advances made by the Lipidomics Cores to develop standardized procedures for lipid analysis and extend the LIPID MAPS database to include the location of lipids in the organelles isolated from macrophages. This will be done with both resting and macrophages activated in vitro by factors involved in macrophage-initiated diseases. In order to accomplish the above goals and objectives, three Specific Aims will be evaluated: (1) Adapt or develop routine procedures for the isolation of macrophage subcellular fractions for lipid analysis by the Lipidomic Cores. The subcellular fractions will include endoplasmic reticulum, Golgi, endosomes, lysosomes, peroxisomes, plasma membrane and its apical and basolateral domains for cells in monolayers, and the inner and outer membranes of mitochondria and nuclei. (2) Determine the changes in lipid composition of macrophage organelles activated by lipopolysaccharide (LPS) and during endocytosis of polystyrene beads in vitro. Both LPS activation and endocytosis albeit of microorganisms occur in inflammation. (3) Determine the changes in lipid composition of macrophage organelles that occur during the different stages of macrophage adhesion to a collagen matrix. The stages are cell attachment, spreading, and migration. Such stages are analogous to those used by macrophages during normal and activated functions in tissues. Accomplishing these aims and making the LIPID MAPS database generated from the results immediately available to others should expedite our understanding of how lipids initiate, regulate and propagate disease.